A Bacillus thuringiensis host strain with high melanin production for preparation of light-stable biopesticides

The bacterium Bacillus thuringiensis produces a crystal protein with insecticidal properties; however, crystal proteins can be damaged by ultraviolet (UV) radiation. The aim of this study was to improve the stability of the insecticidal crystal protein (ICP) by constructing a mutant line that expresses high levels of the UV light-protecting pigment, melanin. BMB181, a B. thuringiensis mutant with high melanin production, was obtained after sub-culturing BMB171 for several generations at 42 °C. The melanin yield by BMB181 (without tyrosine supplementation) reached 8.55 mg/ml. The electroporation efficiency of BMB181 reached 10⁶ CFU/μg when a 6.7-kb foreign plasmid was used. Microscopic and SDS-PAGE analyses revealed that ICP (CryIAc10; GenBank: AAA73077.1), which is highly toxic to Lepidoptera, was synthesized efficiently by strain BMB181. The insecticidal properties of a recombinant line derived from strain BMB181, designated BMB32 (cry1Ac10/BMB181), was tested against the cotton bollworm, Helicoverpa armigera. After UV irradiation for 4 h, BMB32 had a half maximal inhibitory concentration value of 1.37 μg/ml, whereas the control line BMB31 (cry1Ac10/BMB171) had a median lethal dose value of 25.85 μg/ml. These results indicate that the B. thuringiensis mutant is a candidate for industrial scale production of light-stable insecticides.     

A UV Tolerant Mutant of Bacillus thuringiensis subsp. kurstaki Producing Melanin

A UV-resistant mutant (Bt-m) of Bacillus thuringiensis subsp. kurstaki, producing a dark brown pigment, identified as melanin, was studied. Bt-m had higher larvicidity against Heliothis armigera than its parent. Survival of Bt-m spores and their insecticidal activity to irradiation at 254 nm and 366 nm were higher than those of the parent. The only toxic polypeptide produced by Bt-m was Cry1Ac (130 kDa); it lost cry1Aa, cry2Aa, and cry2Ab. Received: 2 April 2001 / Accepted: 14 May 2001     

Antifungal activity of Bacillus thuringiensis strains and their efficacy against the cotton leaf worm Spodoptera littoralis

Bacillus thuringiensis strains that belong to B. thuringiensis, B. kurstaki and soil-isolated B.t. were assessed in the following phytopathogenic: Rhizoctonia solani, that had their mycelial growth decreased after incubation in the presence of the bacterial strains. The bacteria have also pathogenic effect against the insect pest Spodoptera littoralis. The isolate B.t. D-1 and the B.t. kurstaki HD-203 were found to be inhibiting R. solani, the strain B. kurstaki HD-203 displayed the highest percentage of inhibition (64%) and B.t. D-1 showed 49% of inhibition. Antagonistic activity was maintained up to pH 8.5, and the antifungal activity was stable to heat at 70?°C for 1?h. Minimal inhibitory concentrations were 152 and 131?μl/ ml for B.t. D-1 and B. kurstaki HD-203, respectively. The two strains also have high efficacy against S. littoralis larvae, B.t. D-1 gave 70% and the B. kurstaki HD-203 strain gave 80% mortality after seven days of treatment.     

Spectral Characteristics of Near-Infrared Surface Plasmon Resonance

Intrinsic properties of surface plasmons (SPs) excited with Kretschmann configuration were analyzed as a function of wavelength, including the propagation length, the penetration depth, the Goos–Hänchen (GH) shift, and the field enhancement. The calculated results indicate that there exists a critical thickness (t _cr) of the gold layer and that the maximum GH shift occurring exactly at the SP resonance wavelength (λ _R) rapidly varies from positive to negative with changing of the gold layer thickness from t?<?t _cr to t?>?t _cr. The maximum field enhancement happens not at λ _R but at a wavelength smaller than λ _R due to the phase retardation between the transmitted and reflected light. Simulations also reveal that a broadband collimated near-infrared beam can simultaneously excite two SPs with different responses to a refractive index (RI) change: the shorter-wavelength SP able to make a small redshift and the longer-wavelength SP capable of yielding a large blueshift. Only the shorter-wavelength SP was experimentally observed and its RI sensitivity was measured to increase from 3,539 nm/RIU at λ _R?=?707.6 nm to 57,143 nm/RIU at λ _R?=?1,398 nm. The SP at λ _R?=?1,013 nm moved to λ _R?=?1,029 nm in response to the saturation adsorption of bovine serum albumin, and the corresponding surface coverage was determined to be Γ?=?1.565 ng/mm² based on a quasilinear dependence of Γ on the resonance wavelength shift (?λ _R) deduced theoretically. Butyrylcholinesterase adsorption from a dilute solution of 10 nM protein in phosphate buffer solution leads to a redshift of ?λ _R?=?10 nm, corresponding to Γ?≈?0.97 ng/mm².     

Cloning and expression of <Emphasis Type="Italic">mel</Emphasis> gene from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Previous work from our laboratory has shown that most of Bacillus thuringiensis strains possess the ability to produce melanin in the presence of l-tyrosine at elevated temperatures (42 °C). Furthermore, it was shown that the melanin produced by B. thuringiensis was synthesized by the action of tyrosinase, which catalyzed the conversion of l-tyrosine, via l-DOPA, to melanin. In this study, the tyrosinase-encoding gene (mel) from B. thuringiensis 4D11 was cloned using PCR techniques and expressed in Escherichia coli DH5 . A DNA fragment with 1179 bp which contained the intact mel gene in the recombinant plasmid pGEM1179 imparted the ability to synthesize melanin to the E. coli recipient strain. The nucleotide sequence of this DNA fragment revealed an open reading frame of 744 bp, encoding a protein of 248 amino acids. The novel mel gene from B.thuringiensis expressed in E. coli DH5 conferred UV protection on the recipient strain.     

Diversity analysis and characterization of Coleoptera-, Hemiptera- and Nematode-active cry genes in native isolates of Bacillus thuringiensis

Applications to combat non-lepidopteran insects are not as common as applications against lepidopteran insects. The aim of the present work was to isolate and identify Bacillus thuringiensis isolates from soil samples using five approaches, viz., analysis of crystal protein production by microscopy; detection of cry gene content by PCR, SDS-PAGE profiling; cloning and sequencing; phylogenetic analysis; and toxicity testing. Two hundred soil samples were used for isolation of B. thuringiensis and a total of 69 putative isolates of B. thuringiensis that produce parasporal crystalline inclusions were isolated from 5,267 Bacillus-like colonies. A bipyramidal inclusion was predominant in 32.2 % of the B. thuringiensis isolates compared to other shapes. Crystal protein profiling of B. thuringiensis isolates by SDS-PAGE analysis showed the presence of bands of 130, 73, 34, 25 and 13 kDa, among which 50–60 kDa bands were present abundantly. PCR analysis revealed the predominance of Coleopteran-active cry genes in these isolates. Variation in nucleotide sequences, crystal morphology and mass of crystal protein(s) purified from the isolates of B. thuringiensis revealed genetic and molecular diversity. Four strains containing Coleopteran-active cry genes showed higher toxicity against Myllocerus undecimpustulatus undatus Marshall (Coleoptera: Curculionidae) adults when compared with B. thuringiensis subsp. morrisoni pathovar tenebrionis. These results are useful in emphasizing the distribution of cry genes and for prognostication of toxicity, and may contribute to the identification of novel candidate genes for bioengineered crop protection.     

Parasporin 1Ac2, a Novel Cytotoxic Crystal Protein Isolated from Bacillus thuringiensis B0462 Strain

Two novel parasporin (PS) genes were cloned from Bacillus thuringiensis B0462 strain. One was 100 % identical even in nucleotide sequence level with that of parasporin-1Aa (PS1Aa1) from B. thuringiensis A1190 strain. The other (PS1Ac2) showed significant homology (99 % identity) to that of PS1Ac1 from B. thuringiensis 87-29 strain. The 15 kDa (S¹¹³–R²⁵⁰) and 60 kDa (I²⁵¹–S⁷⁷⁷) fragments consisting of an active form of PS1Ac2 were expressed as His-tag fusion. Upon purification under denaturing condition and refolding, the recombinant polypeptides were applied to cancer cells to analyze their cytotoxicities. 3-(4,5-Dimethyl-2-thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide assay revealed that either of 15 or 60 kDa polypeptide exhibited no cytotoxicity to HeLa cells, but they became cytotoxic upon mixed together. Our results suggested that PS1Ac2 was responsible for the cytotoxicity of B. thuringiensis B0462 strain, and that the formation of hetero-dimer of 15 and 60 kDa polypeptide was required for their cytotoxicity.     

Interaction between the entomopathogenic bacterium Bacillus thuringiensis subsp. kurstaki and two entomopathogenic fungi in bio-control of Sesamia nonagrioides (Lefebvre) (Lepidoptera: Noctuidae)

The interactions between the entomopathogenic bacterium Bacillus thuringiensis ssp. kurstaki and two entomopathogenic fungi Beauveria bassiana Balsamo (Vuillemin) (Hypocreales: Cordycipitaceae) and Metarhizium robertsii (Metchnikoff) Sorokin (Hypocreales: Clavicipitaceae) were examined on larvae of Sesamia nonagrioides (Lefebvre) (Lepidoptera: Noctuidae) in 8, 13 and 16 days post-treatment intervals. An overall positive interaction between the pathogens was observed and the larval mortality at 16 days was 56–100 % exposed to M. robertsii combined with B. thuringiensis subsp. kurstaki, whereas B. bassiana combined with B. thuringiensis ssp. kurstaki killed 54–100 % of exposed larvae. After 8 days, in 6 of the combinations, we found an additive relationship between the pathogens, whereas, a negative interaction was observed in 10 of them. In contrast, after 13 days, in 2 of the combinations the positive interaction could be considered as synergistic between pathogens, in 10 as additive, and in only 4 as negative. Finally, after 16 days, in 11 of the combinations we found an additive connection between the pathogens, wheras a negative interaction was seen in 5. Applying both pathogens simultaneously offers a method of Sesamia nonagrioides control that could be more effective than using each pathogen separately.     

Attenuator LRR – a regulatory tool for modulating gene expression in Gram-positive bacteria

With the rapid development of synthetic biology in recent years, particular attention has been paid to RNA devices, especially riboswitches, because of their significant and diverse regulatory roles in prokaryotic and eukaryotic cells. Due to the limited performance and context-dependence of riboswitches, only a few of them (such as theophylline, tetracycline and ciprofloxacin riboswitches) have been utilized as regulatory tools in biotechnology. In the present study, we demonstrated that a ribosome-dependent ribo-regulator, LRR, discovered in our previous work, exhibits an attractive regulatory performance. Specifically, it offers a 60-fold change in expression in the presence of retapamulin and a low level of leaky expression of about 1–2% without antibiotics. Moreover, LRR can be combined with different promoters and performs well in Bacillus thuringiensis, B. cereus, B. amyloliquefaciens, and B. subtilis. Additionally, LRR also functions in the Gram-negative bacterium Escherichia coli. Furthermore, we demonstrate its ability to control melanin metabolism in B. thuringiensis BMB171. Our results show that LRR can be applied to regulate gene expression, construct genetic circuits and tune metabolic pathways, and has great potential for many applications in synthetic biology.     

Silver nanoparticles from Prosopis glandulosa and their potential application as biocontrol of Acinetobacter calcoaceticus and Bacillus cereus

In the present study the characterization and properties of silver nanoparticles from Prosopis glandulosa leaf extract (AgNPs) were investigated using UV–Vis spectroscopic techniques, energy dispersive X-ray spectrometers (EDS), zeta potential and dynamic light scattering. The UV–Vis spectroscopic analysis showed the absorbance peaked at 487 nm, which indicated the synthesis of silver nanoparticles. The experimental results showed silver nanoparticles had Z-average diameter of 421 nm with higher stability (?200 mV). The EDS analysis also exhibited presentation of silver element. Additionally, the different concentrations of AgNPs (25, 50, 75 and 100 mg/mL) showed antibacterial activity against Acinetobacter calcoaceticus and Bacillus cereus. Finally, AgNPs from leaf extracts of P. glandulosa may be used as an agent of biocontrol of microorganism of importance medical. However, further studies will be needed to fully understand the antimicrobial activity of silver nanoparticles obtain from P. glandulosa.     

Decolorization of textile dye RB19 using volcanic rock matrix immobilized <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> cells with surface displayed laccase

A triplicate volcanic rock matrix–Bacillus thuringiensis–laccase WlacD (VRMs–Bt–WlacD) dye decolorization system was developed. WlacD was displayed on the B. thuringiensis MB174 cell surface to prepare a whole-cell laccase biocatalyst by using two repeat N-terminal domains of autolysin Mbg (Mbgn)₂ as the anchoring motif. Immunofluorescence microscopic assays confirmed that the fusion protein (Mbgn)₂–WlacD was anchored on the surface of the recombinant B. thuringiensis MB174. After optimization by a single factor test, L ₉(3⁴)-orthogonal test, Plackett–Burman test, steepest ascent method, and Box–Behnken response surface methodology, the whole-cell specific laccase activity of B. thuringiensis MB174 was improved to 555.2 U L^?1, which was 2.25 times than that of the primary culture condition. Optimized B. thuringiensis MB174 cells were further adsorbed by VRMs to prepare VRMs–Bt–WlacD, an immobilized whole-cell laccase biocatalyst. Decolorization capacity of as-prepared VRMs–Bt–WlacD toward an initial concentration of 500 mg L^?1 of an textile dye reactive blue 19 (RB19) aqueous solution reached 72.36% at a solid-to-liquid ratio of 10 g–100 mL. Repeated decolorization-activation operations showed the high decolorization capacity of VRMs–Bt–WlacD and have the potential for large-scale or continuous operations.     

Chemical changes in the haemolymph ofSpodoptera littoralis [Lep.: Noctuidae] as affected byBacillus thuringiensis

Analyses of the haemolymph of the larvae ofSpodoptera littoralis Boisduval with an amino acid analyser indicated the presence of a group of amino acids, some of which showed an obvious quantitative decrease as a result of treatment withBacillus thuringiensis Berliner such as threonine, serine, asparagine, alanine, valine, methionine, isoleucine, leucine, phenyl alanine and ornithine. Glutamic acid, cystine, β-alanine, γ-butyric acid and histidine contents of the haemolymph of diseased larvae were higher than those in the haemolymph of normal individuals. This increase in the content of some amino acids in infested larvae may be attributed to the possible dissolution of protein crystals ofBacillus thuringiensis. The activity of proteolytic enzymes in the gut may be partly involved in the reaction. B. thuringiensis was found to affect the concentration of some ions of the haemolymph ofS. littoralis. So, a marked decrease in sodium and potassium concentration was observed 4–5 days after treatment withB. thuringiensis. The change in Na⁺/K⁺ ratio in healthy and treated larvae possibly indicate their interference, in the larval toxicity withB. thuringiensis. An obvious decrease in the concentration of magnesium and zinc was also observed in the larval haemolymph after feeding on a diet containing theB. thuringiensis preparations. The pH value of the haemolymph however showed no change after treatment withB. thuringiensis.

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Résumé L'analyse de l'hémolymphe des chenilles deSpodoptera littoralis Boisduval à l'aide d'un analyseur d'acides aminés montre une diminution significative de la quantité de certains acides aminés à la suite d'un traitement parBacillus thuringiensis Berliner, tels que: thréonine, sérine, asparagine, alanine, valine, methionine, isoleucine, leucine, phenylalanine et ornithine. La teneur en acide glutamique, cystine, β alanine, γ acide butyrique et histidine de l'hémolymphe des larves traitées est plus élevée que celle des chenilles témoins. Cette augmentation est peut-être liée à la dissolution des protéines du cristal deB. thuringiensis. B. thuringiensis affecte également la concentration de certains ions dans l'hémolymphe deS. littoralis. On observe une nette réduction du taux de sodium et de potassium 4 à 5 jours après le traitement. Il est possible que cette modification du rapport Na⁺/K⁺ chez les larves malades traduise l'interférence de ces deux ions dans les phénomènes de toxicité deB. thuringiensis. Une diminution nette de la concentration en magnésium et en zinc est également notée dans l'hémolymphe des chenilles après alimentation sur un milieu traité parB. thuringiensis. Le pH de l'hémolymphe n'est pas affecté.

     

Bacillus thuringiensis isolates from soil and diseased insects in Egyptian cotton fields and their activity against lepidopterous insects

The distribution of Bacillus thuringiensis in Egyptian soils of cotton cultivations represented by 11 governorates was studied. This study aimed to isolate indigenous strains that may be potent against some lepidopterous insect pests. Out of 45 isolates, only 10 were effective and they showed high levels of toxicity against the target insects at 500 μg/ml (80% larval mortality or higher). Among these 10 isolates, two isolates were potent against Spodoptera littoralis, two isolates were potent against Helicoverpa (= Heliothis) armigera, seven isolates were potent against Pectinophora gossypiella, but none were potent against Agrotis ypsilon. LC₅₀ and LC₉₀ values and the potency of the B. thuringiensis isolates have been determined. Trials were conducted to isolate B. thuringiensis from diseased lepidopterous insects collected from the same locations, but all isolates showed low potential activity against the target insects. These findings showed promise for the possible use of some indigenous B. thuringiensis strains in the control of lepidopterous pests in Egypt.     

Comparative Sensitivity to UV-B Radiation of Two Bacillus thuringiensis Subspecies and Other Bacillus sp.

Susceptibility of Bacillus thuringiensis spores and toxins to the UV-B range (280–330 nm) of the solar spectrum reaching Earth's surface may be responsible for its inactivation and low persistence in nature. Spores of the mosquito larvicidal B. thuringiensis subsp. israelensis were significantly more resistant to UV-B than spores of the lepidopteran-active subsp. kurstaki. Spores of subsp. israelensis were as resistant to UV-B as spores of B. subtilis and more resistant than spores of the closely related B. cereus and another mosquito larvicidal species B. sphaericus. Sensitivity of B. thuringiensis subsp. israelensis spores to UV-B radiation depended upon their culture age; 24-h cultures, approaching maximal larvicidal activity, were still sensitive. Maximal resistance to UV-B was achieved only at 48 h. Received: 13 December 2000/Accepted: 19 January 2001     

Co-utilization of Crude Glycerol and Biowastes for Producing Polyhydroxyalkanoates

Polyhydroxyalkanoate (PHA) production by Bacillus thuringiensis EGU45 and defined mixed culture of Bacillus spp. were studied by using crude glycerol (CG) and hydrolyzed biowastes as feed material. Hydrolysates from onion peels (OP), potato peels, pea-shells (PS), apple pomace 2% total solids obtained with defined mixed hydrolytic cultures (MHC2) were inoculated with B. thuringiensis EGU45 and defined mixed bacterial cultures (5MC1), which produced PHA at the rate of 40–350 and 65–450 mg/L, respectively. Addition of CG (1%, v/v) to these hydrolysates resulted in 1.8-fold and 4.5-fold enhancement in PHA production from OP by B. thuringiensis EGU45 and 5MC1, respectively. Co-utilization of OP and PS (in 2:1 ratio) supplemented with CG (1%, v/v) by B. thuringiensis EGU45 resulted in 2-fold increase in PHA production in comparison to OP + CG. This co-metabolism of OP and PS also enabled PHA co-polymer production (1300 mg/L), having an enhanced HV content of 21.2% (w/w).     

Chitinase production by <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> and <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>: Their potential in antifungal biocontrol

Thirty bacterial strains were isolated from the rhizosphere of plants collected from Egypt and screened for production of chitinase enzymes. Bacillus thuringiensis NM101-19 and Bacillus licheniformis NM120-17 had the highest chitinolytic activities amongst those investigated. The production of chitinase by B. thuringiensis and B. licheniformis was optimized using colloidal chitin medium amended with 1.5% colloidal chitin, with casein as a nitrogen source, at 30°C after five days of incubation. An enhancement of chitinase production by the two species was observed by addition of sugar substances and dried fungal mats to the colloidal chitin media. The optimal conditions for chitinase activity by B. thuringiensis and B. licheniformis were at 40°C, pH 7.0 and pH 8.0, respectively. Na⁺, Mg²⁺, Cu²⁺, and Ca²⁺ caused enhancement of enzyme activities whereas they were markedly inhibited by Zn²⁺, Hg²⁺, and Ag⁺. In vitro, B. thuringiensis and B. licheniformis chitinases had potential for cell wall lysis of many phytopathogenic fungi tested. The addition of B. thuringiensis chitinase was more effective than that of B. licheniformis in increasing the germination of soybean seeds infected with various phytopathogenic fungi.     

Phototactic behaviour of Eucryptorrhynchus scrobiculatus and E. brandti (Coleoptera: Curculionidae) adults*

Eucryptorrhynchus scrobiculatus and E. brandti (Coleoptera: Curculionidae) are destructive weevils on Ailanthus altissima in China. This study examined phototactic behaviour of E. scrobiculatus and E. brandti in response to eight light-emitting diodes (LEDs) in the laboratory and field. Effects of gender, starvation, and light and dark experience on the phototactic behaviour of the insects were evaluated. The results demonstrated that, the two species of weevil were phototactic insects and most sensitive to violet light (400–405?nm), followed by blue–violet light (420–430?nm). They were less sensitive to red (655–660?nm), white (6000–6500?k), blue–green (470–480?nm), yellow (590–595?nm), blue (450–455?nm), and green (515–530?nm) light. In the light intensity range of 200–1000?lux, the light intensity had no significant effect on the phototatic behaviour of E. scrobiculatus and E. brandti. Phototactic behaviour of the insects was affected by gender. The phototaxis indices of the two species of weevil increased with starvation, reaching a plateau after 2 or 3?d of starvation. The phototaxis indices of E. scrobiculatus and E. brandti were significantly affected by various wavelengths of light following exposure to the light for 3?h or in different dark experience time. In the mark-release-recapture test, the number of E. scrobiculatus and E. brandti adults trapped by violet (400–405?nm) light traps is the largest. The information provided here provides a basis for survey and control of E. scrobiculatus and E. brandti.     

Melanin pigmentation gives rise to black spots on the wings of the silkworm Bombyx mori

Several mutants of the silkworm Bombyx mori show body color variation at the larval and adult stages. The Wild wing spot (Ws) mutant exhibits a phenotype in which the moth has a spot on the apex of the forewing. In this study, we investigated this trait to elucidate the molecular mechanism underlying the color pattern. Microscopy of the black spot of Ws mutants showed that the pigment emerges in the scales of the wing, and accumulation of the pigment becomes strong just before eclosion. We next examined the relationship between the black spot of the Ws mutant and melanin. The spectrophotometry using alkaline extracts from the black spot in the wing showed the highest absorption intensity at 405 nm, which is the absorbance wavelength of melanin. Moreover, inhibition assays for enzymes implicated in melanin synthesis using 3-iodo-l-tyrosine (a tyrosine hydroxylase inhibitor) and L-α-methyl-DOPA (a dopa decarboxylase inhibitor) revealed that treatment with each inhibitor disrupted the pigmentation of the wing of the Ws mutant. On the basis of these results, we analyzed the expression pattern of five genes involved in melanin formation, and found that the expression levels of yellow and laccase2 were increased just before pigmentation, whereas those of DDC, tan, and TH were increased when the apex of the wing turned black. These results showed that melanin pigmentation gives rise to the black spot on the wing.     

Role of Ni-tolerant Bacillus spp. and Althea rosea L. in the phytoremediation of Ni-contaminated soils

In our current study, four nickel-tolerant (Ni-tolerant) bacterial species viz, Bacillus thuringiensis 002, Bacillus fortis 162, Bacillus subtilis 174, and Bacillus farraginis 354, were screened using Ni-contaminated media. The screened microbes exhibited positive results for synthesis of indole acetic acid (IAA), siderophore production, and phosphate solubilization. The effects of these screened microbes on Ni mobility in the soil, root elongation, plant biomass, and Ni uptake in Althea rosea plants grown in Ni-contaminated soil (200 mg Ni kg^?1) were evaluated. Significantly higher value for water-extractable Ni (38 mg kg^?1) was observed in case of Ni-amended soils inoculated with B. subtilis 174. Similarly, B. thuringiensis 002, B. fortis 162, and B. subtilis 174 significantly enhanced growth and Ni uptake in A. rosea. The Ni uptake in the shoots and roots of B. subtilis 174-inoculated plants enhanced up to 1.7 and 1.6-fold, respectively, as compared to that in the un-inoculated control. Bacterial inoculation also significantly improved the root and shoot biomass of treated plants. The current study presents a novel approach for bacteria-assisted phytoremediation of Ni-contaminated areas.     

Susceptibility of the pine processionary caterpillar Thaumetopoea pityocampa (Lepidoptera: Thaumetopoeidae) toward δ-endotoxins of Bacillus thuringiensis under laboratory conditions

A series of natural crystal proteins from B. thuringiensis subsp. Alesti 12–25, caucasicus, galleriae 11–67, galleriae 6–96, kenyae, and shondungensis and spore‐crystal preparations from finitimus 11–66 and from a recombinant strain of B. thuringiensis subsp. kurstaki expressing Cry 1 Ga1 only, were assessed as a toxic agent for the pine processionary caterpillar, Thaumetopoea pityocampa. Some preparations had a thoroughly investigated composition and contained Cry1Aa, Cry1Ab2, Cry1Ab7, Cry1D, Cry1F, Cry 1 Ga1, Cry9Aa, Cry26 crystal proteins, whereas crystals of B. thuringiensis subsp. caucasicus, kenyae, and shondungensis harboured predominantly unidentified toxins distant from commonly used prototypes. Bioassays were based on the simultaneous assignment of each treatment to groups of 20 full sibling first‐instar larvae, obtained from broods of a population from North‐western Italy. The toxin was applied to pine needles by the leaf dipping method and the effect was registered in both feeding inhibition and mortality. B. thuringiensis subsp. caucasicus, kenyae, galleriae 6–96, alesti, and galleriae 11–67 gave the best results in terms of both feeding inhibition and larval mortality. Broods tested in B. thuringiensis bioassays showed a substantial variation in susceptibility to the toxins, suggesting the potential development of resistance in the population.